Bottom Line:
Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide.On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration.The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.

ABSTRACTStructure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic α-helices, (ii) intrinsically disordered peptides, as well as (iii) β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state (19)F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl)-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg) as a reporter group for (19)F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼ 55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.

pone-0099653-g001: CD spectra of the CF3-Bpg labeled TP10 analogs.CD spectra are recorded in the presence of unilamellar DMPC/DMPG (3∶1) vesicles at a P/L ratio of 1∶200. (A) L-epimers and (B) D-epimers are compared with the WT peptide (black line). Analogs with CF3-Bpg in the galanin part are represented by green lines and in the mastoparan part by red lines.

Mentions:
To examine the influence of 19F-labeling on the secondary structure of TP10, we studied the (WT) peptide and the 18 CF3-Bpg labeled analogs by CD spectroscopy in solution. The peptide was added to small unilamellar lipid vesicles composed of DMPC/DMPG (molar ratio of 3∶1) at three different peptide-to-lipid molar ratios (P/L), namely 1∶50, 1∶100 and 1∶200. In these freshly prepared samples, all TP10 analogs have an α-helical conformation similar to the WT peptide at all concentrations tested. CD spectra with a P/L of 1∶200 are shown in Figure 1A and 1B for the L- and D-epimers, respectively. (Data on P/L of 1∶50 and 1∶100 are presented in Figure S1 in File S1). The L-epimers showed no signs of perturbation, neither when the label was incorporated in the N-terminal galanin part, which is colored in green in this and subsequent figures (including residue Ile8, as will become obvious from the data below), nor in the C-terminal mastoparan part (colored in red). We may thus conclude that the TP10 analogs labeled with L-CF3-Bpg are essentially unperturbed and can be used for the subsequent 19F-NMR structure analysis. Rough secondary structure estimation for the WT peptide by deconvolution of the CD data revealed a helix fraction of around 56%, as shown in Table 1. This corresponds to a stretch of 12 amino acids that are helically folded, while the remaining part of the sequence is predominantly disordered.

pone-0099653-g001: CD spectra of the CF3-Bpg labeled TP10 analogs.CD spectra are recorded in the presence of unilamellar DMPC/DMPG (3∶1) vesicles at a P/L ratio of 1∶200. (A) L-epimers and (B) D-epimers are compared with the WT peptide (black line). Analogs with CF3-Bpg in the galanin part are represented by green lines and in the mastoparan part by red lines.

Mentions:
To examine the influence of 19F-labeling on the secondary structure of TP10, we studied the (WT) peptide and the 18 CF3-Bpg labeled analogs by CD spectroscopy in solution. The peptide was added to small unilamellar lipid vesicles composed of DMPC/DMPG (molar ratio of 3∶1) at three different peptide-to-lipid molar ratios (P/L), namely 1∶50, 1∶100 and 1∶200. In these freshly prepared samples, all TP10 analogs have an α-helical conformation similar to the WT peptide at all concentrations tested. CD spectra with a P/L of 1∶200 are shown in Figure 1A and 1B for the L- and D-epimers, respectively. (Data on P/L of 1∶50 and 1∶100 are presented in Figure S1 in File S1). The L-epimers showed no signs of perturbation, neither when the label was incorporated in the N-terminal galanin part, which is colored in green in this and subsequent figures (including residue Ile8, as will become obvious from the data below), nor in the C-terminal mastoparan part (colored in red). We may thus conclude that the TP10 analogs labeled with L-CF3-Bpg are essentially unperturbed and can be used for the subsequent 19F-NMR structure analysis. Rough secondary structure estimation for the WT peptide by deconvolution of the CD data revealed a helix fraction of around 56%, as shown in Table 1. This corresponds to a stretch of 12 amino acids that are helically folded, while the remaining part of the sequence is predominantly disordered.

Bottom Line:
Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide.On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration.The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.

ABSTRACTStructure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic α-helices, (ii) intrinsically disordered peptides, as well as (iii) β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state (19)F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl)-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg) as a reporter group for (19)F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼ 55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.